A fundamental understanding of how the sigma-1 receptor is regulated at the protein level, and how this impacts its function, is still missing. This is important because the sigma receptors represent a novel class of proteins that when activated by psychostimulants, such as cocaine and methamphetamine, contribute to both short- and long-term adaptations in the brain. Our long-term goal is to elucidate the regulatory mechanisms controlling activation of sigma receptors as a prerequisite to fully understanding sigma receptor function. The overall objective of this R03 application, which is a first step toward attainment of our long-term goal, is to fully characterize the sigma-1 receptor and its "signaling complex." The central hypothesis of this application is that the sigma-1 receptor utilizes a network of interacting proteins to translate ligand binding events into biological action and this signaling network is subject to regulation by post-translational protein modifications and differential protein-protein interactions. The rationale for the proposed research is that it will be possible to study sigma-1 receptor regulation of additive behaviors more thoroughly once we understand how its signaling is controlled. Since the sigma-1 receptor is associated with various biological processes, this knowledge will potentially lead to successful strategies for targeting structurally (and functionally) diverse isoforms specifically. The proposed research is relevant to NIH's mission because it will expand our basic knowledge and potentially improve the human health. Two specific aims will be pursued in order to test our central hypothesis and accomplish the objective of this application: 1) Identify sigma-1 receptor post-translational modifications in the presence and absence of ligands;and 2) Identify sigma-1 receptor interacting-proteins in the presence and absence of ligands. We will accomplish this by using tailored purification schemes and specific labeling strategies in conjunction with optimized HPLC gradients and state-of-the art mass spectrometry instrumentation. The proposed research is significant because it will lead to a better fundamental comprehension of a novel class of membrane bound receptors and expand our understanding of how the receptor is regulated by drugs of abuse. PUBLIC HEALTH RELEVANCE: Sigma-1 receptors, potentially important therapeutic targets for drug addiction and abuse, will be comprehensively characterized in the proposed studies. The proposed research is relevant to public health because the results are expected to lead to a better understanding of how these proteins can be manipulated to treat substance abuse.